MTG16 contributes to colonic epithelial integrity in experimental colitis

Abstract

Objective: The myeloid translocation genes (MTGs) are transcriptional corepressors with both Mtg8(-/-) and Mtgr1(-/-) mice showing developmental and/or differentiation defects in the intestine. We sought to determine the role of MTG16 in intestinal integrity.

Methods: Baseline and stress induced colonic phenotypes were examined in Mtg16(-/-) mice. To unmask phenotypes, we treated Mtg16(-/-) mice with dextran sodium sulphate (DSS) or infected them with Citrobacter rodentium and the colons were examined for ulceration and for changes in proliferation, apoptosis and inflammation.

Results: Mtg16(-/-) mice have altered immune subsets, suggesting priming towards Th1 responses. Mtg16(-/-) mice developed increased weight loss, diarrhoea, mortality and histological colitis and there were increased innate (Gr1(+), F4/80(+), CD11c(+) and MHCII(+); CD11c(+)) and Th1 adaptive (CD4) immune cells in Mtg16(-/-) colons after DSS treatment. Additionally, there was increased apoptosis and a compensatory increased proliferation in Mtg16(-/-) colons. Compared with wild-type mice, Mtg16(-/-) mice exhibited increased colonic CD4;IFN-γ cells in vehicle-treated and DSS-treated mice. Adoptive transfer of wild-type marrow into Mtg16(-/-) recipients did not rescue the Mtg16(-/-) injury phenotype. Isolated colonic epithelial cells from DSS-treated Mtg16(-/-) mice exhibited increased KC (Cxcl1) mRNA expression when compared with wild-type mice. Mtg16(-/-) mice infected with C rodentium had more severe colitis and greater bacterial colonisation. Last, MTG16 mRNA levels were reduced in human ulcerative colitis versus normal colon tissues.

Conclusions: These observations indicate that MTG16 is critical for colonocyte survival and regeneration in response to intestinal injury and provide evidence that this transcriptional corepressor regulates inflammatory recruitment in response to injury.

Keywords: Colitis; Crohn's colitis; Helicobacter pylori; Helicobacter pylori infection; Helicobacter pylori—pathogenesis; IBD; IBD basic research; IBD—genetics; Mtg16; angiogenesis; bacterial pathogenesis; cancer; carcinogenesis; colon carcinogenesis; colorectal cancer; colorectal diseases; cytokines; dextran sulphate sodium; experimental colitis; gastrointestinal pathology; gene expression; immunity; inflammatory bowel disease; inflammatory mechanisms; intestinal T cells; matrix metalloproteinase; ulcerative; wound-healing.

Figure 1

Myeloid translocation gene-16 (MTG16) regulates colonocyte proliferation. (A) H&E stained Mtg16^−/− or WT colons. (B) In situ TUNEL staining. Results presented as the number of TUNEL labelled cells/crypt, 20 crypts/mouse counted (p=0.86) (n=9 mice/genotype). (C) Representative photomicrographs of Ki67 staining in WT or Mtg16^−/− colonic crypts. Graph at right shows comparison between Mtg16^−/− and WT Ki67 counts per crypt, with an average of 50 crypts/mouse counted (**p<0.01, n=4 mice/genotype). (D) FITC-dextran permeability assay (*p<0.05) (n=6 WT, n=7 Mtg16^−/−).

Figure 2

Immunophenotyping of Mtg16^−/− colons reveals increased innate and adaptive immunity. (A) Immunophenotyping using flow cytometry of WT or Mtg16^−/− colons (WT (n=6) or Mtg16^−/− (n=6) colons). (B) Representative density plots for CD4 IFN-γ-producing cells are shown (cell percentages displayed in corners). (C) Increased IFN-γ production by CD4 cells in Mtg16^−/− colons. (D) mRNA expression from Mtg16^−/− colons presented as fold change from WT (dashed line, SD shown, ***p<0.001). (E) Multi-analyte profiling using the Luminex platform demonstrating increased IL-17, IL-4 and MIP-1β in Mtg16^−/− colons (*p<0.05, **p<0.01, ***p<0.001).

Figure 3

MTG16 is involved in the regulation of colonic injury. (A) WT (n=8) or Mtg16^−/− (n=7) mice were treated with 4% dextran sodium sulphate (DSS) (w/v) ad lib for 7 days. Animals were sacrificed upon achieving 20% weight loss. Body weights (mean body weight ± SD) are shown at days 0, 4 and 7 as per cent change from baseline (day 0 weight). (B) Kaplan–Meier survival curves. (C) Per cent difference in colon length. (D) ‘Swiss-rolled’ colons stained with H&E. (E) Histological injury score using a multi-point scale as described in the Methods section. (F) Citrobacter rodentium-colitis model H&E stained sections. (G) Histological injury scores as described in the Methods section. (H) Colonic bacterial colonisation (*p<0.05, **p<0.01, ***p<0.001).

Figure 4

MTG16 contributes to epithelial restitution after injury. Mtg16^−/− (n=14) or WT (n=17) mice were treated with 3% dextran sodium sulphate (DSS) (w/v) or water ad lib for 4 days followed by recovery on water for 3 days prior to euthanising. (A) Body weights (mean body weight ± SD) are shown at days 0, 4 and 7 as per cent change from baseline (day 0 weight). (B) Per cent difference in colon length. (C) H&E stained representative sections of Mtg16^−/− or WT Swiss-rolled colons. Images at right represent boxed area from 40× images at left. (D) Quantification of colonic injury represented as an aggregate histological injury score. (E) Ki67 immunohistochemistry. (F) Representative activated caspase-3 immunohistochemistry (left) after 4 days of 3% DSS treatment (Bar denotes 10 μM). Quantification of activated caspase-3 staining (right) (n=5 per genotype) (*p<0.05, **p<0.01, ***p<0.001: DSS-treated compared with control (H₂0) mice, ¶p<0.05, ¶¶¶p<0.001: Mtg16^−/− vs WT DSS, §§p<0.01: Mtg16^−/− compared with WT control mice).

Figure 5

Adoptive transfer of WT marrow into Mtg16^−/− mice is incapable of rescuing the Mtg16^−/− colonic injury phenotype. Mtg16^−/− (n=6) or WT (n=6) mice were lethally irradiated and rescued with isogenic WT marrow. Three months post-transplantation mice were treated with 3% dextran sodium sulphate (DSS) ad lib for 4 days followed by 3 days of recovery on water. Weights are shown in (A). H&E stained Swiss-rolled sections are shown in (B). Histological injury score was tallied using a multipoint scale and is presented in (C) (*p<0.05, ***p<0.001: DSS compared with Control, ¶ p<0.05: Mtg16^−/− DSS compared with WT DSS).

Figure 6

Exacerbated innate and adaptive immune responses in Mtg16^−/− mice following dextran sodium sulphate (DSS) injury. Mtg16^−/− (n=8) or WT (n=8) mice treated with water or 3% DSS (w/v) according to our injury-repair model (4 days 3% DSS ad lib followed by 3 days of water recovery). (A) Flow cytometric analysis using haematopoietic lineage-specific antibodies from WT (n=8) or Mtg16^−/− (n=8) colons. An unpaired two-tailed t test indicated that there was a significant increase in Gr1⁺, F4/80⁺, CD11c⁺, MHCII expressing CD11c⁺, CD4⁺ and IFN-γ expressing CD4⁺ cells in Mtg16^−/− colons. (B) Representative cell plot histograms of MHCII expressing CD11c and IFN-γ expressing CD4 populations. (C) RNA expression levels measured by qRT-PCR on Mtg16^−/− colons presented as fold change from WT (dashed line). Error bars represent SD, **p<0.01, ***p<0.001. (D) Multi-analyte profiling using the Luminex platform revealed increased INF-γ, IL-17, TNFα, IL-4, and IL-13 and chemokines MIP-1β and KC in Mtg16^−/− colons. (E) Epithelial-specific KC mRNA expression levels (*p<0.05, **p<0.01, ***p<0.001; unidimensional ANOVA).

Figure 7

MTG16 is underexpressed in moderate to severe ulcerative colitis (UC). Mtg16 mRNA levels were determined using qRT-PCR on UC biopsy specimens. (A) Mtg16 mRNA levels expressed as negative ddCt in normal (n=10) versus UC (n=35) samples. This represents a 2.8-fold decrease in expression. (B) Subset analysis of Mtg16 expression in UC (normal n=10, quiescent-mild n=14, moderate-severe n=21). This represents a 4.2-fold decrease in Mtg16 mRNA levels in moderate-severe UC samples. **p=0.005, Wilcoxon rank sum, in comparison with control samples (normal).